POPULATION STUDIES ON FIVE GENERA OF INTESTINAL BACTERIA OF SWINE During Normal Conditions of Health During Artificially Induced Hog Cholera by Mohan Singh Purohit A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health 1953 POPULATION STUDIES ON FIVE GENERA OF INTESTINAL BACTERIA OF SWINE DURING NORMAL CONDITIONS OF HEALTH DURING ARTIFICIALLY INDUCED HOG CHOLERA by Mohan Singh Purohit AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied S ci e n c e in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public H e a l t h Year Approved 1953 Population studies of five genera of intestinal bacteria of swine during health and artificially induced hog cholera were made. During health, the lactobacilli were highest in number from six to seven weeks of age. The number of lacto­ bacilli decreased in healthy pigs from eight weeks of age up­ ward. There was a dramatic decrease or disappearance of lactobacilli from the intestinal tract once the animals had developed clinical symptoms of hog cholera. The coliforms, which are usually regarded as harmless parasites, were least numerous during health irrespective of age differences. The average number of coliforms ranged be­ tween 13 to 37 millions per gram ;of fecal material. The number of coliforms increased many times once the animals under experiment became sick and developed diarrhea. The number of enterococci was higher than that of the coliforms but lower than that of the lactobacilli up to seven weeks of age during health. In pigs from the age of eight weeks upward the enterococci were most numerous. The aver­ age number of enterococci was b2 to 8^ millions per gram of fecal material. There were no significant differences between their numbers during infection. Salmonella choleraesuis isolated from healthy as well as diseased swine. was During the entire period of experiment S. choleraesuis was isolated seven times. S. cubana was isolated from the intestinal tract of in­ fected pigs for the first time (3 cases). 2 Among sixty-four cultures of enterococci isolated from fecal samples the predominant group was Streptococcus fecalis. DEDICATED TO Dr. H. J. Stafseth, an inspiring teacher and sincere friend, with an ideal philosophy for directing graduate studies. ACKNOWLEDGMENTS The author wishes to express his sincere feelings of gratitude to Dr. H. J. Stafseth, Head of the Department of Bacteriology and Public Health, under whose inspiration, guidance, and constant encouragement it was possible to carry out this work and prepare this thesis. He expresses thanks to Dr. H. W. Dunne and Dr. W. N. Mack for their help in the investigation and the facilities provided in the experimental laboratory. The author wishes to take this opportunity to express his gratitude to all staff members concerned at Gamp Detrick for materials. Thanks are expressed to Dr. R. A. Runnells, Dr. W. 0. Brinker, Dr. C. A. Lawson, and Dr. W. D. Baten for their guidance, helpful suggestions and assistance. Grateful acknowledgment is made Dr. and Mrs. Ralph D. Barner for their encouragement all along the period of study in this country. The writer shall always remain obliged to his friend, Roopraj Purohit and family, to Michigan State College, and to the people who support this abode of learning for their financial aid in the form of tuition scholarship and assistantship during the study in this country. MOHAN SINGH PUROHIT candidate for the degree of DOCTOR OP PHILOSOPHY Final examination, June 17, 1953? 8:00 A.M., Room 101, Giltner Hall. Dissertation: Population Studies on Five Genera of Intesti­ nal Bacteria of Swine. During Normal Conditions of Health During Artificially Induced Hog Cholera Outline of Studies Major subject: Veterinary Bacteriology Minor subjects: Surgery and Medicine, Animal Pathology Biographical Items Born, January 1, 1921+, Ghenri, Jodhpur Division, Rajasthan Province, India. Undergraduate Studies, Jodhpur, i9*+2-¥+: Binar Veterinary College, 1 9 ^ - 1+7. G.B.V.C. Graduate Studies, Michigan State College, 19^9-1953• Experience: Veterinary Assistant Surgeon and Livestock Inspector, 19^7-^9j Research Assistant, Department of Surgery and Medicine, Michigan State College, 1952-53• Member of the Society of Sigma Xi TABLE OF CONTENTS Page INTRODUCTION .......................................... 1 HISTORICAL REVIEW ..................................... 3 EXPERIMENTAL PROCEDURE ................................ 21 RESULTS AND DISCUSSION ................................ 37 SUMMARY AND CONCLUSIONS ............................... 73 BIBLIOGRAPHY .......................................... 7^ INTRODUCTION The importance of the bacterial flora of the intestines has long been recognized. It has been suggested that animal life would be impossible without the micro-organisms found in the digestive tract. In order to gain an understanding of the role of the microflora of the gut, Schottelius (1), in 1899? raised normal and germ-free chickens on diets which today would be considered totally inadequate. The slow growth and abnormal­ ities of the chicks were such that he concluded that normal chicks could not be raised germ-free. Further, he came to the conclusion that the function of the normal nonpathogenic flora was to aid in preparing ingesta for absorption, to stimulate the intestinal wall and to promote peristalsis, to oppose the establishment of the pathogens, and to aid in defense of the host. Cohendy (2), in 1912? reported his results on germ-free work. His diets were protein free and vitamin deficient. was unable to rear sterile chicks beyond five weeks. He Neither Cohendy nor Schottelius was concerned with the systematic classification of the bacteria. In their studies quantita­ tive assays were not made. Creekmur (3), in 1922, studied the intestinal flora of rats on normal diets and on one deficient in vitamin A. The 2 feces of animals on deficient diet became dry and hard with distinct decrease in the total number of bacteria. The ad­ dition of vitamin A in the form of a few drops of cod liver oil daily produced normal conditions. In germ-free studies the workers did not attempt to classify or enumerate the bacteria of the intestinal tract. Enrichment and selective media, by which these workers could have broken down these groups of intestinal bacteria, were not available. As the intestinal bacteria play a significant role in health and disease, the writer took the opportunity to study these micro-organisms in swine under normal condition of health and in animals artificially challenged with hog cholera virus. The purpose of the study was to determine which of the intestinal organisms (coliforms, enteric streptococci, lacto­ bacilli, or salmonellae) increase or decrease in numbers while the animal is suffering from hog cholera. 3 HISTORICAL REVIEW Relation of bacteria to life. debated question. This has been a much Nuttal and Thierfelder C1*), in 1895, (5) 1896, and (6) 1897, reared guinea pigs which had been deliver­ ed by Caesarian section under aseptic conditions. The animals increased in weight, but not as regularly as normal pigs. From these data the authors concluded that bacteria were not necessary for normal development. Levin (7), in 1899, found that the intestines of most of the arctic animals were sterile. He drew no conclusions, but his work is of interest because normal growth took place without bacteria. Wollman (8) concluded from the experiment on flies that bacteria were not necessary for normal de­ velopment. Kianzine (9)? in 1918, produced data concerning germfree chicks which he thought weakened the argument of Cohendy. Kianzine believed that bacteria were of great aid in di­ gestion; they carry on analytic and synthetic processes which are of much value to the host. He observed that when guinea pigs were made to breathe sterile air and eat sterile food they were greatly weakened, even after a few days. The bad results from the deprivation of bacteria were thought to be due to reduced oxidation and accumulation of leucomains in the if body. Loeb and Northrup (10) attempted to determine the ability of flies to synthesize their body proteins without the aid of micro-organisms. Larvae grown on sterile media (banana) did not do well, while those placed on sterile yeast culture developed normally. It was thought that yeast was essential for this species of fly. Studies of different microflorae. The earliest studies of the flora of different animals and birds were not quanti­ tative in the sense that there were no tallies of populations of different types; e.g., coliforms, enteric streptococci, aerobes and anaerobes. The enrichment and differential media available today had not been developed. Gage (11), in 1911, studied the intestinal flora of chickens. He found that Nocardia asteroides, Bacillus mv- coides. Bacillus subtilis, Micrococcus, pseudomonads, and Escherichia coli predominated, but there was variation due to environmental conditions and the age of the chickens. He also estimated that 60 percent of the bacteria in the intesti­ nal tract of the chickens were gram negative in their stain­ ing characteristic. Some difficulty was encountered in dis­ tinguishing bacteria from debris, much of which was gram posi­ tive. While working with rats, Hull and Rettger (12), in 1917, and Torry (13), in 1919, established beyond doubt a re­ lationship between the type of flora predominating in the in­ testinal tract and the type of food ingested. Mens and 5 Rochlin (I1*-), in 1929, while working with chickens, found the same bacterial species in all parts of the intestine, the difference being in quantity. This uniformity of the flora was explained on the basis of acid production which suppressed putrefactive bacteria and their associated decomposition processes. The flora producing lactic acid was: Strepto­ coccus fecalis, Escherichia coli, and Lactobacillus plantarum, the latter being the most prominent anaerobe. Emmel (15), in 1930, found a decrease in the number of E. coli in 20 chickens suffering from enteritis. Salmonella pullorum was invariably in the intestines of the chickens suffering from pullorum disease, fowl typhoid, and paratyphoid. In their work on the study of the influence of diet on the distribution of bacteria in the various parts of the gastrointestinal canal of rats fed on stock diet, Porter and Rettger (16), in 19^0 , showed that, whereas the normal bac­ terial flora of the young white rats is comparatively simple in the upper segments of the alimentary tract, it becomes increasingly more abundant in the lower sections of the small intestine. Windblad (17), in 19*+!, and Mitchell and Isbell (18), in 19^2, while working on rats, showed a relationship between the type of micro-organisms predominating in the intestinal tract and the type of diet given. During a study of the bac­ terial flora of the feces of rats, Miller (19), in 19*+5, ob­ served that the number of organisms present varied from rat to 6 rat and in the same animal from day to day. McGinnis (20), in 1950? stated that growth acceleration in chicks, when anti­ biotics are fed, might be a result of either: 1) reduction in the total number of bacteria in the intestinal tract; 2) syn­ thesis of vitamins by bacteria; 3) the inhibition of either pathogenic or toxin producing bacteria. Groschke (21), in 1950, suggested that antibiotics stimulate the growth in chicks by changing the intestinal microflora from undesirable to de­ sirable ones. Couch et al. (22), in 1950, reported an in­ crease in the total count of bacteria in the feces of chicks when penicillin was included in the ration. Wahlstorm et al. (23), in 1951? reported that penicillin had no significant effect on the number of coliform bacteria present in the feces of baby pigs fed a "synthetic" milk diet. Coliform Group in Health and Disease Escherichia and Aerobacter Escherichia coli and Aerobacter aerogenes were isolated by Escherich from the feces of infants and described in 1886 (2*+). Since that time the organisms have been found in the intestinal tract of all vertebrates. Hadley (25), in 1910, isolated B. coli from the liver and heart blood of sick chickens affected with a cholera-like di­ sease. High temperature and yellowish diarrhea were the 7 symptoms observed upon examination; the liver was hyperemic and soft and the spleen enlarged and hemorrhagic. The inflam­ mation extended through the entire intestine. Johnes (26), in 1918, studied three cases of mastitis from two of which E. coli was isolated and from the third A. aerogenes was cultured. In two of the cases the milk con­ tained numerous white floccules and in the third it had the appearance of hemolysed blood. Gaiger and Dalling (27), in 1921, reported that lamb dysentery is caused by a B. coli type which invades the body from the intestines. Palmer and Baker (28), in 1922, found that Bacterium coli communis was the causative organism of an infectious enteritis of chickens, ducks, and turkeys in northern and central Delaware. Gaiger and Dalling (29), in 1923, reported that lamb dysentery is caused by an anaerobe of the Clostridium welchii type and a coli type conjointly. Kinsley (30), in 1928, described B. coli communis, B. suipestifer, and other closely related bacteria commonly found in scours in pigs. He assumed that these enteric organisms had attained pathogenicity. Tunicliff et al. (31)> in 1932, were able to produce fatal dysentery in lambs by feeding the cultures of several varieties of Escherichia, although several strains failed to produce dysentery. From this observation they came to the conclusion that "under favorable conditions certain strains of bacteria, principally Escherichia which are normally present in the intestines may produce disease". 8 Davis (32), in 1938, encountered colibacillosis in chickens in Maryland. Upon autopsy the livers were about 50 percent larger than normal, mottled, and contained numerous small white spots. The heart was slightly enlarged and the peri­ cardial sac was markedly distended with amber colored fluid. Twisselmann (33)? in 1939? reported that seventeen birds of a well isolated group of 250 pullets were attacked by a rapidly fatal disease of which he stated E. coli was the etiological agent. Bunyea and MacDonald (3*+)? in 19*+!? studied the patho- genecity of E. coli for turkeys and considered it responsible for young poult mortality. Murphy (35)? in 19^3» described 70 cases of bovine mam­ mary gland infection where coliform organisms were the chief invaders. For three years a study of milk and udders of a herd of about 120 Guernsey and Holstein cattle was made. The infections were observed from one day to twenty-two months and the degree of irritation ranged from negligible to severe acute mastitis. Sixty-nine of the infecting organisms were studied and the results were as follows: *+1 (59.*+ percent) were A. aerogenes type; 18 (26.1 percent) were intermediate types; and 10 (1^.5 percent) were E. coli types. Hjarre and Warmby (36), in 19^5? described a specific granuloma-like condition in chickens which was found along the intestinal tract. The lesion was considered to be a typical form of tuberculosis, but a capsulated B. coli was the only organism isolated. The investigators subsequently found that 9 such an organism is the etiological agent. According to Udall (37), in 19^7, it was recognized that various influences such as season, sanitation, and exposure contribute to the cause of diarrhea. Great importance was attached to the action of bacteria which are normally present in the lower intestine, especially B. coli. Osborne, Witter, and Hitchner (38), in 19^7, studied chronic colibacillosis of fowls. The organism which they studied was proved to be very pathogenic for guinea pigs, rabbits, and chickens. Durant and MacDougle (39), in I9V 7, found E. coli in the blood of adult fowl affected with an occular form of fowl paralysis. When 1 ml amounts of the blood taken from the affected birds were injected into day old chicks, 8^- of 85 chicks died. Hagen (^0), in 1951, stated that the organisms of the colibacillus type are generally regarded as causative agents of the disease commonly called calf scours. It is a known fact that the tissues of animals suffering from this disease are teeming with E. coli shortly before and after death. He further stated: Bacterium coli is a normal inhabitant of the lower bowels of warm blooded animals. It usually is absent from the intestines of fish and other cold blooded animals. Few or none are found in the stomach or anterior part of the bowel. Carnivora and omnivora usually harbor the organ­ ism in greater abundance than herbivora. The feces of cows and horses frequently show very few. Enterococci in Health and Disease The term enterococcus has long been used by French work­ ers to designate a group of streptococci that normally occurs in the intestine of man. The most important of these organisms is Streptococcus fecalis. It is known that this organism occurs in the intestines of several domestic animals as well as of man. Enterococci occasionally have been found in patho­ genic processes but generally are looked upon as harmless to man and animals. They are characterized by great hardiness. Initial isolation. Since its use by Thiercelin (^1) in 1899 and (*+2) in 1902, the term Enterococcus has had somewhat variable and hazy meanings. In some cases the name has been applied rather specifically as a synonym for Streptococcus fecalis. Thiercelin’s findings led him to believe that enter­ ococci are causal agents in certain diarrheal biliary in­ fections and the cause of appendicitis. MacCullum and Has­ tings (*+3), in 1899> studied a case of acute endocarditis and isolated a new species which they named Micrococcus zvmogenes and described it as follows: This micrococcus is very small, occurs mainly in pairs, sometimes in short chains, gram positive, lique­ fies gelatin slowly and is specially characterized by its behavior in milk which it acidifies, coagulates and liquefies. It produces milk curdling ferments, each of which is separable from the bacterial cells. It remains viable for months in old cultures and is tolerably re­ sistant to the action of heat and antiseptics. The micrococcus is pathogenic for mice and rabbits causing abscesses or general infections. 11 Streptococcus fecalis was named by Andrews and Horder (¥f), in 1906, who gave an excellent description of the organ­ ism in view of the means for studying bacteria then at hand. The organism was described as nonhemolytic, having a strong reducing action on neutral red, coagulating milk and ferment­ ing the dissacharides, salicin and mannitol, but not inulin and usually not raffinose. The fermentation of mannitol was considered very characteristic. The work of Andrews and Horder on intestinal streptococci was verified by a number of subsequent workers. Winslow and Palmer (*+5), in 1910; Fuller and Armstrong (*+6), in 1913; and Broadhurst (^7), in 1915. According to Donaldson (^8), in 1917, the French workers thought the enterococci to be constant inhabitants of normal intestines and commonly found in the small intestines of infants three days after birth. Further it was observed that the enterococcus was pathogenic for rabbits. Oppenheim (*+9), in 1920, studied the human fecal streptococci and observed that the mannite fermenting, nonhemolytic ones were the characteristic predominant types found in the feces of normal individuals. Hemolytic streptococci were found only occasion­ ally in the stools of normal, healthy people. Ayers and Johnson (50), in 192b, examined 33 human fecal cultures and found that the predominant species was Str. fecalis. Sherman and Wing (51), in 1935 and 1937, described a new species of hemolytic streptococcus which was tolerant 12 to heat and desiccation. On the basis of its physiological characteristics, Streptococcus durans was related by these workers to the enterococci, though it was not known to be of intestinal origin. Recent studies of the hemolytic strepto­ cocci of the human intestine by Sherman (52) have shown that this organism commonly occurs in human feces. Although a num­ ber of species of hemolytic streptococci may be isolated from human feces, so far as present information extends, Str. dur­ ans and Str. zvmogenes are the only intestinal types which may be properly designated as "hemolytic enterococci". Common Habitat of Enterococci Brige (53)? in 1905? isolated a micrococcus from labora­ tory animals corresponding morphologically and culturally to that isolated by MacCullum and Hastings in 1899? with the ex­ ception that this organism was nonpathogenic for laboratory animals. Andrews (5*+), in 1906, stated that streptococci can­ not grow and multiply for any length of time outside the human body. Broadhurst (55)? in 1915? was of the opinion that strep­ tococci occurred less commonly in soils and water than most of the literature of that time implied. Alston (56), in 1928, studied 50 strains of streptococci from the alimentary tract of man, dog, and rat. Thirty-five percent of them were des­ cribed as streptococci, oval in shape, and occurring in pairs or short chains, nonhemolytic and capable of fermenting manni- 13 tol. Smith and Sherman (57), in 1938, studied the hemolytic streptococci of human feces and found that the commonest hemo­ lytic streptococcus of the human intestine is Str. zvmogenes. with Str. durans following next. Porch (58), in 191*!, demon­ strated that the enterococci were the predominant group of streptococci recovered from the genitourinary tract. Winter and Sanhozer (59), in 19*+6, reported that, x^hile streptococci were present in all samples of human and animal feces tested, these organisms were never found in virgin soils or soils from wooded areas. Mallmann and Litsky (60), in 1951, using dextrose azide broth as enrichment medium, could not isolate enterococci from soils which x^ere not treated with sewage. They also stated that other than coliform organisms, the enter­ ococci were the only organisms found in sewage that could be used as indicators of fecal pollution. Salmonella Choleraesuis in Health and Disease This organism was isolated and described by Salmon and Smith (61) in 1885 and was the first of the paratyphoid organ­ isms to be recognized. It is because of this fact that these organisms are known under the generic name of Salmonella. The authors believed the organisms to be the cause of the de­ structive and prevalent disease known as hog cholera, but later work demonstrated that this is not the case, hog cholera being caused by a filtrable virus. Nevertheless, this organ- 1*+ ism plays an important role in porcine pathology, principally as a secondary invader in virus diseases. Trawinski (62), in 1917, made bacteriologic examinations of the intestinal contents of 500 butchered hogs and isolated not less than 26 strains of microbes which apparently could be classified as colon-typhoid intermediates. Dorset (63), in 1921, stated that the presence of the virus of hog cholera vastly increases the susceptibility of hogs to suipestifer in­ fection. Moussu (6b), in 192*f, believed that the causative microbes live in a given environment as saprophytes and as such may exist in the digestive tract of swine. Faulty sani­ tary conditions, indigestion, irritation of the digestive or­ gans by feed of inferior quality, or even slight intoxications are apt to reduce the normal defensive powers of the body to such an extent that the infective agent begins to multiply in profusion and becomes progressively more virulent with an in­ fectious enteritis as a result. Gl&sser (65), in 1927, recognized damp, stuffy, dirty stables, faulty feeding, deficiency of minerals and protein in the ration, lack of paddocks and atmospheric influences, such as excessive humidity, as important predisposing factors for Salmonella infection. That the hog cholera bacillus is not constantly asso­ ciated with hog cholera was also shown by Uhlenhut and asso­ ciates (66), in 1929, when they made careful bacteriological examination of 178 cases of hog cholera among which S. sui- 15 pestifer could be demonstrated in 76 animals. Birch et al. (67), in 1937, and Chick et al. (68), in 1938, did not be­ lieve that all swine enteritis is caused by the invasion of salmonellae, but the nutrition of the pig plays a very impor­ tant part in the etiology of this disease. These workers found that severe deficiency could be produced in young pigs fed a low protein diet made up largely of corn, and that the deficiency could be prevented by the addition of niacin. Dale (69), in 19*+2, stated that although normal hogs harbor this organism, it has been found in a sufficient number to indicate that there may be a carrier type in which the microbe is present without causing appreciable injury. Stafseth, Lisa Neu, and Sholl (70), in 1 9 ^ , isolated S. choleraesuis from a pup which had died. Cultures were made from the heart, liver, spleen, and kidneys. From all these sources almost pure cultures of a Salmonella-like organism were obtained. Studies of morphology, staining, cultural and biochemical characteristics of this organism indicated that it was S. choleraesuis. variety Kunzendorf. They were also able to isolate the same organism from the vaginal swab ob­ tained from the mother of the pup. Schofield (71) 5 in 1 9 ^ ? stated: In making a diagnosis of salmonellosis based upon the constant isolation of salmonellae, it is important to remember that S. choleraesuis and other members of this group are not infrequently present in the in­ testine of normal swine; that such animals when attack­ ed by a virus septicaemia, as in hog cholera, may suffer severe intestinal damage from activated and virulent 16 salmonellae. It is quite possible, in fact alreadyrecognized by some investigators, that in other enteric diseases such as swine dysentery, salmonellae may appear either as secondary invaders or proliferate due to alteration in environment. In hogs, necrotic enteritis was produced by Rasmussen, et al. (72), in 1 9 ^ , by administering S. choleraesuis. A more se­ vere reaction was secured when the culture was mixed in the feed than when given as a drench. Difference in virulence of the culture could have been responsible for the difference in reaction, but this reason is unlikely according to these authors. Many hogs died from the initial attack of the di­ sease regardless of nutritional regimen, but during the re­ covery period nicotinic acid and some principle or principles present in liver, but not in sufficient quantities in yeast, were of benefit by promoting increased weight gains and de­ creasing the length of the recovery period. The authors be­ lieved that a composite of known B vitamins may give better response than nicotinic acid alone in the recovery phase of the disease. Schofield, loc. cit., in 19i+1+, further came to the con­ clusion that S. choleraesuis is responsible for damage to the mucous membrane which later is frequently invaded by the normal inhabitant of the intestine, Actinomyces necrophorous. The pigs between the ages of eight to sixteen weeks appear to be particularly susceptible to this infection. Though de­ fective nutrition may be a factor in predisposing to in­ fection, poor sanitation and overcrowding are conducive to rapid spread of infection. 17 Doyle and Walkey (73), in 19*+6, proved experimentally that feeding of viscera from pigs affected with what was known as "necrotic enteritis" to cholera vaccinated shoats produced no clinical symptoms of disease. However, when parts of the same viscera were fed to cholera susceptible pigs, one-half of them showed lesions of hog cholera and enteritis. This would indicate that the virus of hog cholera produced enter­ itis. They also concluded that enteritis resulting from hog cholera was different from that produced by salmonellae. Lucke and associates (7*+), in 19^7, and (75) in 19^8, showed that the level of protein in the ration was directly related to the severity of the experimentally produced niacin de­ ficiency. Pigs fed a ration containing only 15 percent pro­ tein developed severe deficiency symptoms, whereas those fed a ration containing 19 percent protein developed only mild symptoms of the disease. In much of the work involving the relationship of niacin to necrotic enteritis the procedure was to feed large doses of S. choleraesuis in culture broth to the pigs. It was observed that niacin had little effect in either preventing or curing the resulting enteritis. Edwards and his associates (7&), in 19^8, discussed the finding of Salmonella types in swine and stated that "in the writer's experience salmonellae are much more likely to be isolated from cases of acute enteritis than from those with chronic necrotic enteritis". Lucke and his co-workers (77), in 19*+9, fed the pigs intestinal material from which Salmonella 18 newport and Salmonella tvphlmurlum had been isolated. The animals showed no particular temperature rise nor diarrhea, while a pig in another group when fed S. choleraesuis showed temperature rise to 105° F for one day and failure to eat. The pig returned to normal after 72 hours. Lactobacilli in Health and Disease The genus Lactobacillus constitutes one of the most wide­ ly distributed groups of known micro-organisms. The group as a whole is primarily fermentive in character, and displays a marked degree of tolerance for acids. Members of this genus vary widely in their physiological activities and in their habitat. Some are characteristically associated with milk and dairy products; others are intimately associated with raw cereals and vegetables; and others are of oral and dental origin. Still others are primarily intestinal types. The in­ testinal types constitute the material for the present study. The genus Lactobacillus is grouped by Bergy and co-workers (78) in the family Lactobacteriaceae which includes the strep­ tococci and the pneumococci. Bergey lists about 15 species, only two of which are parasitic, Lactobacillus acidophilus and Lactobacillus bifidus. The lactobacilli have not been successfully indicated as pathogens within the customary mean­ ing of that term. On the contrary, they have been praised for beneficial effects in the vagina and intestine of human hosts. 19 DiJderlein (79), in 1892, reported that vaginal secretion of many pregnant women contained a group of micro-organisms consisting of large numbers of bacilli and some yeasts. He also found that in women whose vaginal secretion was less acid the flora was more varied and there was a higher incidence of puerperal disease. He thought that lactic acid produced by vaginal microbes limited the growth of other micro-organisms in the vagina and thus protected the area against infection. Moro (80), in 1900, isolated an organism from the feces of breast fed infants to which he gave the name Bacillus aci­ dophilus. Moro's organism was, as he supposed, derived from the mother’s breast and was found in the mouths and stomachs of infants as well as in the intestinal contents. He stated that it was not a single species but a group of closely re­ lated forms which preferred an acid medium for growth. Weiss (81), in 190^, noted that when large quantities of milk were ingested a marked increase in organisms of the lactobacillus type was observed. Tissier (82), in 1908, in a study of the intestinal flora of infants observed that, at the age of about three days, breast fed babies developed an in­ testinal flora in which one organism was predominant. This organism had, in general, the staining reactions and morpho­ logical appearance of the lactobacilli. As Tissier noted the presence of many bifurcated forms, he called it Bacillus bifidus. There was a considerable controversy between Tissier and Moro as to the predominant organism in breast fed babies 20 and it was finally settled by Moro’s admission that L. bifidus occurred in far greater numbers. However, both investigators concluded that L. acidophilus became predominant in the feces of babies whose chief food was cow’s milk. Rettger and associates (83) showed that the feeding of milk or lactose in large amounts to white rats and human subjects brings about the transformation of the intestinal flora to one in which the lactobacilli predominate. Morishita (81*), in 1929, Curran et al. (85), in 1933, and Gillespie and Rettger (86), in 1938, suggested that oral lactobacilli are distinct from the intestinal ones, whereas Sherman at (87), in 19^+0 , were unable to distinguish the oral lactobacilli from intestinal types. Infection with lactobacilli. Howitt (88), in 1930, re­ ported that in rabbits the intravenous injection of large doses of lactobacilli was followed by development of joint lesions with extensive mucopurulent exudate containing numer­ ous polymorphonuclear leucocytes and macrophages, and occasion­ al gram positive rods. Jay et al. (89), in 1932, noted the development of sterile abscesses following subcutaneous in­ oculations of lactobacillus vaccines in both human subjects and rabbits. 21 EXPERIMENTAL PROCEDURE Media used. In the choice of media, it was thought de­ sirable to have a set of media in which the determination could be made under identical conditions. Considering the large number of organisms to be estimated, a dilution count method was deemed most practical and was employed for nearly all organisms except salmonellae and lactobacilli. For sal- monellae, different selective media were used to observe frequency of presence in the healthy and virus challenged pigs. For lactobacilli, the pour plate method was adopted. It was considered necessary that one medium give a total count of the viable bacteria, and that the selective media allow estimation of the relative numbers of each of the individual groups of bacteria. The total count. means of several media. The total coupt of aerobes was made by Evenson (90), in 19*+6, studying the rodent flora, used a liver infusion broth with a chunk of liver settled at the bottom to permit the anaerobes to grow also. Dhanda (91) > in 19*+7j and McClure (92), in 19^9 > while studying the intestinal flora of rats, used the same medium. Tryptose glucose yeast extract agar (T.G.E.) was employed by Shapiro and Sarles (93), in 19^9* Elam and Gee (9^)j in 1951, used liver infusion broth for total bacterial (aerobic) count. 22 The writer found that tryptose glucose yeast extract agar with fresh liver extract added to it ^tfas more suitable. The liver extract enhanced the growth so the total number of organisms was higher. tMedia for Coliform Organisms (Escherichia coli and Aerobacter aerogenes) Coliform bacteria are of historic importance, particular­ ly because they were reported by early workers as being the dominant flora in the gut and also because many animal nu­ trition studies suggested that the suppression of coliform bacteria was related to vitamin deficiencies. Bile or its salts were extensively used because of their marked selectiv­ ity for growth of coliform organisms. Jackson (95)? in 1907? recommended the use of lactose broth containing bile for elim­ ination of noncoliform gas producers. MacConkey (96), in 1908, used bile salt agar with neutral red, while Rector (97)? in 1913? used dried whole bile. It was found, however, that the concentration necessary for the desired inhibition of gram positive organisms was somewhat inhibitory to the c o n ­ forms as well. Domnik and Lauter (98), in 1929? used a methy­ lene blue-brom creosol purple combination as a confirmation for coliform organisms in water. Stark (99)? in 1936? evalu­ ated the use of formate ricinoleate broth for the detection of coliform organisms in milk. Mallmann and Darby (100) in 23 their studies on the detection of the coliform organisms in water used lauryl tryptose broth. They also found that eosin methylene blue agar and brilliant green bile broth could not be used if it was desired to confirm all positive presumptive tubes which contained coliforms. Evidence to support their statement came from the fact that they were able, in many cases, to isolate coliform organisms from lauryl tryptose positive presumptive tubes which they had failed to confirm by the use of eosin methylene blue agar or brilliant green bile broth. By early workers, the E. coli medium of Hajna and Perry (101) was used as a selective medium for the coliform group. It has been reported that the bile salts in this medium in­ hibit the growth of fecal streptococci and spore formers. Faulkner (102) showed that the female hormones diethylstilbestrol, hetoestrol, and stilboestrol are much more selective­ ly inhibitory for gram positive than for gram negative organ­ isms. Chapman (103) discussed the remarkable selectivity of sodium alkyl sulfate (called Tergitol 7). He claimed that the only gram negative organisms inhibited by this substance were the members of the genus Proteus. According to Naghski, et al. (10^), quercentin is inhibitory to certain gram posi­ tive organisms at a concentration of 0.075 to 0.1 mg per ml, the action being much less on gram negative group. Rittenberg and Silliker (105), in testing penicillin against several organisms, found that coliform organisms could 2k withstand 50 units per ml, while Cl. welchii and enteric strep tococci were inhibited at 30 units. Vaughn et al. (106) described a buffered boric acid lactose medium for enrichment and identification of E. coli. The writer used the lauryl tryptose broth prepared accord­ ing to the formula of Mallmann and Darby, loc. cit., as a pre­ sumptive test medium to determine the presence of coliform organisms. Medium for Enterococci Bagger (107), in 1926, used sterile ox-bile with one per­ cent peptone to grow fecal streptococci. Fleming (108), in 1932, reported that fecal streptococci will grow in a concen­ tration of 1:15,000 of potassium tellurite which is inhibi­ tory to gram negative bacteria. Mallmann (109), in 19^0, re­ ported on a medium containing sodium azide which was found useful in estimating the number of streptococci in sewage as it was found to support the growth of these bacteria while in­ hibiting the coliform group. Hajna and Perry, loc. cit., published the formula for another selective medium which was almost an exact duplicate of the medium suggested by Mallmann; but the former workers used an incubation temper­ ature of ^5° C for growth and the production of acid in this medium was stated to be almost complete evidence of the presence of Str. fecalis. 2? Chapman (110), in 19^6, perfected a medium for the isolation of fecal streptococci. This is the mitis-salivarius agar. Enterococci produce a dark blue or black, slightly raised colony about 1 mm in diameter. The writer chose azide-dextrose medium (Difco), which is primarily a result of the studies of Mallmann and Seligman (111) in 195^. For further confirmation, ethyl violet azide broth, developed by Mallmann and his associates (112) in 1952, was used. For a complete test for enterococci, brain heart infusion agar, brain heart infusion broth (incubated at b5° C), litmus milk, and tryptose phosphate broth with 6.5 percent Nacl were used. Media for Isolation of Salmonella For isolation of Salmonella, the following media were used: For the purpose of enrichment, the media most favored are sodium tetrathionate broth and selenite broth. favored the use of sodium tetrathionate broth. The writer The credit for discovering the usefulness of a tetrathionate broth for enriching typhoid and the paratyphoid organisms is ascribed to Mueller (113) who demonstrated clearly that it inhibited coliforms and permitted the paratyphoids to grow. Further demonstrations of the usefulness of this medium were made by Johnes (ll1*), in 1936, and Ruys (115)» in 19^0. After 12-16 hours of incubation of the fecal sample in tetrathionate 26 broth, MacConkey's agar, and Shigella Salmonella agar (S. S. agar), plates were streaked. S. S. agar. This medium was developed by Difco Labora­ tories to provide differentiation of the lactose fermenters from lactose nonfermenters and to give the maximum growth to coliform organisms without restriction of the growth of the pathogenic gram' negative bacilli. Shigella. Salmonella, and other organisms not fermenting lactose form opaque, trans­ parent and translucent uncolored colonies which generally are smooth. Lactose fermenters which are not inhibited are gener­ ally recognized by the formation of red color in the colony. Some coliforms do not show definite red color, being pink or nearly colorless. Some proteus and salmonella types produce black centered colonies. Bismuth sulfite agar. The early history of the develop­ ment of bismuth sulfite agar may be found in the reports of Wilson and Blair (116), in 1926. These workers clearly demon­ strated the superiority of this type of medium over other media in the isolation of S. typhosa from feces, urine, and sewage. Cope and Kasper (117)? in 1937? increased their find­ ings of typhoid organisms from 1.2 to 16.8 percent among food handlers. Green and Beard (118), in 1938, used successfully bismuth sulfite agar in their studies on the survival of E. typhosa in sewage treatment processes. This medium so success­ fully inhibited sewage organisms that their interference was negligible. 27 Kligler's iron agar. Bacto-Kligler's iron agar is pre­ pared with phenol red as an indicator of the production of acid, and ferrous sulfate as an indicator of HgS production. The dextrose and lactose fermenters are also differentiated. The writer used this medium as one means of isolation of Sal­ monella in fecal samples. Bacto-methvl red Voges Proskaur medium (M.R.V.P.). This medium was used for performance of the M.R.V.P. tests in dif­ ferentiation of Salmonella from other organisms. are M.R. positive and V.P. negative. Salmonellae This medium was also used in differentiation of the coli-aerogenes group. Bacto-urea broth concentrate. This medium was used to differentiate Salmonella and Proteus. Salmonellae are urea negative and could be checked by this medium as well as with M.R.V.P. and indol tests. Bacto-tryntone. Bacto-tryptone in one percent concen­ tration is specified in "Standard Methods for the Examination of Water and Sewage" (119), for the performance of the indol test. This medium was used along with the above mentioned media for isolation of salmonellae. Media for Lactobacilli A large group, not well defined as to the nutritional significance in the gut, is the lactic acid group which in­ cludes the enterococci, coliforms as well as lactobacilli. These organisms grow in anaerobic as well as aerobic media. 28 Tissier (120), in 1899? used glucose infusion agar in veillon tubes for the growth of lactobacilli. Torry (121), in 1917? employed a procedure in which he used a liver infusion glucose agar to which 10 percent rabbit blood was added. Mickle and Breed (122), in 1925? reported the use of tomato juice in culture media for lactobacilli. Later Kulp and White (123)? in 1932, described a modification of the original medi­ um which gave them relatively high quantitative counts. To­ mato juice agar which has been used as a highly selective medium for lactobacilli has the disadvantage that it is not entirely selective for lactobacilli even at an acid reaction. Since yeasts, molds, streptococci, etc., may occur with vary­ ing frequency and this interferes with successful isolation and enumeration of lactobacilli. Evenson et al., loc. cit.. used an acetic acid medium for growing lactobacilli, be­ lieving that a low pH would suppress the growth of other organ­ isms. Dhanda, loc. cit., and McClure, loc. cit.. also used this medium. The writer used a selective medium developed by Morrison et al. (12^) in 1951- This medium gave luxuriant growth of lactobacilli. 4 n The Ingredients of Lactobacillus Medium Trypticase 10 grams Yeast extract ............... 5 11 6 " Ammonium citrate ............ 21 " Glucose 20 " 1 " 25 " Sorbitan mono-oleate Sodium acetate hydrate Acetic acid ................. 1.32 ml Salt solution ............... 5 Agar ........................ 15 grams Distilled water to 1 liter Final pH: Composition of Salt Solution MgSO^, 7H20 ..... 11.5 grams MnSOLj., .2H20 .... FeSO^, 7H20 ..... ” 2.b " 0.68 " Distilled water to 100 ml 30 Animals used* Six- to nineteen-week-old New Hampshire, Durock Jerseys, and Poland China pigs were used for this ex­ periment. These pigs were shipped from the farms to the ex­ perimental station in a healthy condition. Their temperature was recorded before exposing them to hog cholera virus. the same time fecal material was collected. At The animals then were kept in lots in different experimental rooms with free access to fresh water and feed. During the period of the ex­ periment all of the animals were on the same kind of diet. Collection of fecal samples. The fecal material was collected in a sterile container by manipulating the anal area. Care was taken to avoid contamination. The samples were brought to the laboratory within an hour and were kept in a refrigerator until used. Preparation of the fecal material. No common or standard procedures are given by previous workers for assaying the in­ testinal flora. Harrison and Hansen (125), in 19^5^ used sterile sand when grinding fecal material of rats. Shapiro et al. (126), in 19^9, and Evenson et al., loc. cit.. used glass beads in shaking bottles to disperse the fecal clumps. The glass beads were the writer1s choice in preparing most of the fecal samples for serial dilutions. Under aseptic conditions, the writer weighed one gram of the fecal material and transferred it to a 99 ml water blank bottle con­ taining a few glass beads to facilitate uniform suspension 31 following vigorous and thorough shaking (routinely 200 times). This suspension (1-100) of the fecal material was further di­ luted in decimal series to 10-11. If the fecal material was hard, a one gram portion was placed in a sterile mortar and ground with sterile sand. This mixture was poured into a sterile dilution blank and the volume made up to 100 ml be­ fore subsequent tenfold dilutions were made. Portions of each dilution (starting from 1-one million to 1-one hundred billion) were introduced into various tubes containing selective and differential media. Total count of aerobes. One ml of the fecal material from each of the serial dilution blanks was poured into a Petri dish and 15 to 20 ml of tryptose glucose yeast extract agar (T.G.E.) was poured to make pour plates. These Petri dishes, thus inoculated, were incubated for 2b to *+8 hours and the colonies were counted by a Quebec colony counter. All along the experiment this method was followed for total aer­ obic bacterial count. In the beginning of the work plain T.G.E. agar was used, but later on when liver extract was add­ ed to the same medium the total bacterial count increased greatly, which might be due to the many growth stimulating factors which liver contains. Coliform counts. Lauryl tryptose broth prepared accord­ ing to the formula of Mallmann and Darby, cited before, was used as a presumptive test to determine the presence of coli­ form organisms. A tenfold serial dilution of fecal material 32 in sterile distilled water was made up to lO"11 and one ml of each dilution was added to 10 ml of broth. After W-8 hours of incubation at 37° C, the highest dilution showing gas formation was used to indicate the number of coliform organisms present. To confirm the presence of the aerobacter group, eosin methyl­ ene blue agar plates were streaked from the tubes showing the growth of coliforms. I.M.V.I.C. tests were also performed for the confirmation of Aerobacter and Escherichia. Enteric streptococci counts. One ml of each dilution of fecal material prepared as described for the coliforms was added to 10 ml of dextrose azide broth for the determination of streptococci. It was presumed that the positive tubes, after 2k hours incubation at 37° C, indicated the presence of streptococci. azide broth. Further confirmation was made on ethyl violet When tubes other than those showing turbidity at 2k hours were positive at *f8 hours, transfers were also made into ethyl violet azide broth. It was necessary to use four standard loopfuls (diameter 5 mm) to accomplish the transfers. The highest dilution in which turbidity could be observed after *+8 hours of incubation of ethyl violet azide at 37° C was used to estimate the number of enterococci present. Isolation of single colonies of enteric streptococci. method used for isolation of enteric streptococci was the general procedure described by Sherman, loc. cit. A small amount of culture material from the second highest posi­ tive ethyl violet azide dilution was used to streak a brain The 33 heart infusion agar plate. After 2b hours of incubation at 37° C, a few typical single isolated colonies of enteric strep­ tococci were transferred to brain heart infusion broth, tryp­ tose phosphate broth with 6.5 percent salt, and litmus milk. All cultures were incubated at 37° G, except the brain heart infusion broth which was incubated at b5° C. Readings were made after ^8 hours and 72 hours of incubation, and also after 96 hours in the case of litmus milk. Turbidity in the brain heart infusion broth and tryptose phosphate broth containing 6.5 percent salt was considered as a positive test for the presence of enteric streptococci independent of the reaction in milk. The reactions in litmus milk were recorded as partial reduction, or partial or complete coagulation for further study. The enteric streptococci isolated from fecal samples were separated in the following species: Str. fecalis, Str. liciuefaciens. Str. zvmogenes. and Str. durans. The i- dentification was made according to the method proposed by Sherman, loc. cit., in 1937 (see Table 1) and Bergev's Manual. loc. cit.. Resistance of enterococci to 6.5 percent sodium chloride was considered as a significant test for enter­ ococci, hence all cultures growing in sodium chloride tryptose phosphate broth were further identified as follows: Brain heart infusion broth (stock culture) Blood a^ar plates Gelatin Sugars TABLE 1 Different Characteristics of Enterococci* • • Species Str. fecalis Acid production from Milk : Gelatin Hemolysis.liquefaction curdled:ArasMal- :Su- :Lac- :Gly- jMan- :Sor• :binose stose :crose stose :cerol:nitol ibitol • - Str. liciuefaciens Str. zvmosenes • / Str. durans ♦Bacteriological Reviews, 1:80, 1937* £ £ / / £ / / £ / / / — / £ / £ / / / - / / / / / - - 35 Lactobacilli counts. One ml of each dilution of fecal material prepared as described for coliforms and enterococci was placed in Petri dishes and 15-20 ml lactobacillus medium was poured into each plate. A uniform mixture of fecal ma­ terial and the medium was obtained by gentle rotation of the plates. hours. These plates were incubated at 37° C Tor *+8 to 72 The colonies were counted, using a Quebec colony counter. Isolation of salmonellae. To freshly prepared tetra­ thionate broth 0.25 ml iodine solution was added (6 grams of iodine, 5 grams of potassium iodide in 20 ml of water) per 10 ml of the medium. Nearly one gram of fecal material was placed in this medium and it was then incubated for 12-16 hours at 37° C . - After this incubation period the following procedure was adopted: Tetrathionate broth a loopfull of material streaked on SS agar MacConkey plates (incubate 2b hours 37° C colorless colonies picked Kligler1s iron agar (H2S7', alkaline slant, acid butt) Indol, M.R.V.P. and urea tests Sugars 36 When these tests were completed and the organism in question was suspected to be Salmonella, the culture was sent to the Michigan Department of Health laboratories for final confir­ mation and typing. Bismuth sulfite agar plates were also inoculated with fecal material for isolation of salmonellae, but later on this medium was discontinued as the writer was quite satis­ fied with the media and methods mentioned before. Microscopic Examination Throughout the experimental period, smears were prepared from the cultures from time to time and gram staining was done to confirm the microscopic characteristics of the organ­ isms under study. 37 RESULTS AMD DISCUSSION Comparison of Total Aerobic Plate Counts. Coliforms Enterococci, and Lactobacilli During Health As expected, the total counts of the bacteria were always higher than those of the different species during health and disease, irrespective of age differences (Tables 2-22). can be observed in Figures 1, 2, 3, and h-. This Evenson and co- workiers, l o c . cit., while working on the intestinal flora of white rats, also noticed the same results. Since the animals were exposed to hog cholera virus soon after the first fecal cample was taken, no further samples could be obtained for comparison of day to day variations. By an analysis of variance, it was shown that there were no significant differ­ ences between arithmetic averages of these counts (Table 2). The coliforms, which are usually regarded as harmless parasites, were least numerous during healthy conditions ir­ respective of age differences or conditions under which these pigs had been raised. Table 22 shows that the average coliform counts ranged between 13 to 37 millions per gram of fecal material. Such differences in bacterial counts were also observed by White (127), in 19^2, and Driesens (128), in 1952. By analysis of Six tfeeks old pigs 550 500 Plate cot ats (aero bic) Coliforms Enterococ ci Lactobacilli 4-00 Organisms in millions 35.0 300 250 200 100 Oth Pig. 1 8th 12th l6th Days (Period of infection) Variations in infestinal bacterial cotints in six weeks old pigs after infection with hog cholera virus. 38 Seven weeks old pigs 39 IPlate cot nts (aero Coliforms Enterococ ci Lactobacilli 900 850 800 750 650 600 550 Organisms in millions 700 4oo 350 300 200 150 100 50 **ir 0 Oth Pig. 2 4th 8th 12th 16th Days ( Period of infection) Variations in intestinal bacterial counts in seven weeks old pigs after infection with hog cholera virus. Sixteen weeks old pigs 40 550 500 X 31ate cou Lts(aeroM( Joliform s hiterococ ji !jactohaci! .11 450 4oo 1 .. . . . / 350 \ .V 300 250 200 150 • 100 • */ / •/ / •/ ■ t 50 L-x / / __ > •/ / / • i — i--------- 1---------- 2— s.— a— e—^---------^ \— -------------- --------------—* ---* --- * ~ * — o 0th 3 - M -- A- 4th 8th 12 th l6th Days (Period of infection) Variations in intestinal bacterial counts in sixteen weeks old pigs after infection with hog cholera virus. Pigs of 6-19 weeks of age 550 5oo Plate co Coliforms Bnterocoec LactobaciL 450 400 350 300 250 200 150 100 50 o 6 '. 7 Pig. 4 8 10 12 Age m , weel Variations in intestinal "bacterial counts in healthy pigs from six to nineteen v/eeks of age. b2 variance, it was f o u n d that there was no significant vari­ ation due to difference in age. The number of enterococci was higher than that of the coliforms but lower t h a n that of the lactobacilli up to seven weeks of age during good health. From eight weeks on­ ward the enterococci were most numerous, the coliforms were next, and the lactobacilli were least numerous. The average number of enterococci was *+2-8*+ millions per gram of the fecal material. A n analysis of variance showed that the num­ ber of enterococci (6 8 -8*+ millions) was significantly higher in pigs six to eight weeks of age than in pigs ten, twelve, sixteen, and nineteen weeks of age (*+2-66 millions). Radison (129), in 1952, w h i l e working with calves, found higher enterococci counts i n may be presumed that the first few weeks after birth. It since the enterococci are lactose fer­ menters they could g r o w luxuriantly in the milk diet with high lactose content and acid medium. Although the total number of enterococci had decreased after the pigs had reach­ ed the age of eight weeks, they did not decrease to the level of lactobacilli, due to age differences, but remained near the same level. The number of lactobacilli was highest at six to seven weeks of age so far a s individual genera were concerned. How­ ever, there was no significant difference among the average counts of the pigs s i x to eight weeks of age, nor among those of the ten to n i n e t e e n weeks old pigs. This was confirmed **3 by an analysis of variance. The high lactobacilli counts ob­ served. in the pigs while nursing their mothers was to be ex­ pected. The milk with its high lactose content has been known since its early studies by Hull et al. (130), in 1917 > and Cruickshank (131), in 1938, to favor the development of an aciduric flora and depress the growth of coliforms. Figures 1, 2, 3 5 and and Table 22 show a decrease in the numbers of lactobacilli in healthy pigs from eight weeks of age and upward. This could have been due to the fact that the composition of the stock diet did not offer readily available energy for the growth of lactobacilli. In other words, a large amount of indigestible material and high protein present in the stock diet may have had its effect in this respect. RATION FED TO THE PIGS DURING EXPERIMENT Ground corn 60 Parts Ground oats Meat scrap .... 10 " Soybean oilmeal 1*+ " 3 ” Mineral ..... Irradiated yeast (Vitamin Dg) 120,000 units Per 100 pounds M-f- Comparison of Total Aerobic Plate Counts, Coliforms, Enterococci and Lactobacilli in Swine Challenged with Hog Cholera Virus After taking the first fecal sample, the pigs were in­ jected intracranially or intramuscularly with hog cholera virus. The animals were kept in different experimental pens. During the assay of the fecal sample after every fourth day, it was noticed that there was no significant difference in the average of the total aerobic plate counts. The numbers remained high during the entire period of infection (Tables 2, 3, 10, and 17). At the same time the growth curves dur­ ing the infected stage corresponded to the trend of the curve of the coliforms, as shown in Figures 1, 2, and 3« The number of the coliforms increased many times when the animals under experiment became sick and developed diar­ rhea. This may be observed in Tables *+, 5> 11 j and 18. In most of the cases the coliform index remained high during the entire period of infection, and there was little tendency to decrease. There were significant differences between the daily average counts and between the weekly average counts at the one percent level. The increase of coliforms during the period of infection and diarrhea might be due to the de­ crease of lactobacilli and, therefore, less competition for nutriments; there might be some unknown condition favoring their multiplication. As for enterococci, there were no significant differ­ ences between the averages of the total numbers caused by in­ fection, diarrhea, or constipation, as shown in Tables 6 , 7» 12, and 19. It may be presumed that this was due to their ability to survive in adverse environment, as shown by the work of Sherman, loc. cit., Table 23. The decrease or disappearance of the gram positive aciduric organisms from the intestinal tract, once the pig had developed the clinical symptoms, as shown in Tables 8, 13, and 20 and Figures 1, 2, and 3> was probably due to decreased food intake during the period of infection or due to the absence of utilizable carbohydrates, or the alkaline reaction of the medium. While working on the intestinal bacteria of rats, Porter et al. (132), 19^0, also observed similar results. According to these workers, fasting the rats for a period of two days brought about only slight change in the character of the bacterial flora of the intestinal tract. However, when the food was withheld for five days, a very marked transfor­ mation took place. Aciduric organisms of the L. acidophilus type were no longer present in the different sections of the alimentary canal, while the coliform bacteria, which ordi­ narily were observed to be restricted to the ileum and cecum, appeared throughout the small intestine and constituted a large portion of the flora. In this respect it is interest­ ing to note that Rougentzoff (133) observed that when rabbits were allowed to fast there was an absence of an acidophilic 1+6 flora in the intestines but an increase in the number of anaerobes. Also, Poppens (13^), in 1921, found that E. coli increased in the duodenum of dogs when the animals were allowed to fast. Presence of Salmonellae in the Intestinal Tract of Swine During Health and Disease Isolation of S. choleraesuis. The writer x^as interest­ ed to know how often S. choleraesuis could be isolated from the healthy as well as diseased swine. S. choleraesuis, which is regarded as a normal inhabitant of the intestinal tract, according to Runnells (135)? Van Es (13&), and Dale, loc. cit., was isolated infrequently. During the entire period of the experiment, S. choleraesuis was isolated only seven times. S. choleraesuis may have been present in other animals, but isolation was not possible because it may have been outnumbered by other intestinal organisms, such as enter­ ococci, coliforms, lactobacilli, Pasteurella suiseptica, etc. So, when one wants to isolate S. choleraesuis, even on a selective medium, the chances of isolating it from fecal ma­ terial become meager. On the other hand, when the animals had developed infection this organism became a secondary in­ vader and entered the general circulation. In the opinion of the writer, it was because of this that early workers, such as Dorset et al. (137), in 190^, frequently were able to iso­ late S. choleraesuis from the blood of infected pigs but not 1+7 from the intestinal contents. As this study was not concern­ ed with S. choleraesuis in the general circulation, no attempt was made to isolate it from the internal organs. The presence of S. choleraesuis may produce enteritis in swine, but when both the virus and S. choleraesuis are present it is still not proved which one is the real causa­ tive agent of necrotic enteritis. work needs to be carried out. On this problem further Healthy germ-free pigs should be made available and infected with virus and S. choleraesuis separately. It also may be presumed that in hog cholera in­ fection there is a disturbance in host-parasite balance, so that when the natural resistance of the host has declined, S. choleraesuis may invade the tissues through the necrotic areas present in the intestinal tract. Isolation of S. cubana. S. cubana was isolated from pigs (numbers 659, 666, and 667) for the first time. Accord­ ing to Seligman, et al. (138), this organism was first iso­ lated by Curbelo in 19^5 in Havana during an outbreak of diarrhea among chicks. The organism was found in feces and in some instances in the heart blood of affected chicks. chicks were imported from Florida in good health. human infection has been reported. These No case of No symptoms developed as a result of feeding the culture, but the mice died within 20 hours when the culture was injected subcutaneously. It was quite interesting to observe that pigs (numbers 665, 666, 667, 668, 690, and 692) from which salmonellae were *f8 isolated, showed no marked degree of pathological changes in the digestive tract after the period of infection with the hog cholera virus. However, on the other hand, pigs 659, 6*4-69 6*+7 , and 689 which were harboring salmonellae had developed severe hemorrhagic gastritis and enteritis with a few necrotic foci. Their submaxillary lymph nodes also revealed peripher­ al hemorrhage. E. Goli in the Blood Stream During the experimental period, E. coli was isolated from the internal organs (heart blood, liver, kidneys, and spleen) on MacConkey agar plates, eosin methylene blue agar plates, and in lauryl tryptose broth. The isolation of E. coli was correlated with the changes in the digestive tract. On post-mortem examination these animals revealed either ne­ crotic gastroenteritis or petechiae. So far as the patho­ genicity is concerned, the writer is unable to say whether E. coli plays any significant role. But this much can be said, most of these pigs had gastritis or necrotic enteritis. As there was a break in the surface epithelium of the intestinal tract, E. coli had made its way into the general circulation and thus was found in the heart blood, liver, kidney and spleen. McBride (139), in 1931*, while working on an acute enter­ itis in young pigs due to infection with the colon group, ^9 isolated E. coli from the internal organs in every case in which enteritis had developed. Hagen, loc. cit., is of the opinion that E. coli escapes from the digestive tract into the blood stream and is disseminated to all parts of the body about the time of death. Predominant Subgroups of Enterococci in Feces Sixty-four cultures of enterococci isolated from fecal samples taken from healthy pigs, on brain heart infusion agar, were classified by acid production in different sugars, lique­ faction of gelatin and production of hemolysis on blood agar and reduction of litmus milk in 21+-^-8 hours. Most of the organisms of this group were Str. fecalis, as shown in Table 2b. The writer's results agree with those of Ayers and Johnson (1^0) who examined 39 human fecal cultures of entero­ cocci and found that the predominant species was Str. fecalis. They also concluded that Str. fecalis was similar to, if not identical with, Str. lactis. Ritter and Treece (1^-1), in 19*+8, isolated 79 strains of streptococci from swimming pools. were classified as Str. fecalis. Fifty-two (65.8 percent) These were confirmed by the Lancefield technique and. were further classified as type D. TABLE 2 Average Plate Counts (Aerobic) in Millions Age of nigs in weeks Days : t 6 s • 7' : 16 1 356 502.6 2^9.5 >+ 36*+ ^73.5 b2b.5 8 5+65 631.6 251.8 12 312 685.0 522.5 TABLE 3 Plate Counts (Aerobic) in Millions Serial: Pig no . : no. 6 weeks"old pigs Before : After infection infection: 1 st dav : ^th dav : 8th dav :12th dav:loth dav 1 1+9)+ 280 760 5+70 170 2 1+95 3>+2 2*+2 320 600 3 h-96 1+00 980 890 310 !+ b97 1000 160 900 0 5 513 170 250 270 280 6 51>+ 225 800 1000 200 7 515 9^0 210 / 8 525 115 215 313 - 9 526 186 306 318 - 10 528 276 528 260 / 11 512 115 200 250 - 12 527 225 1+00 130 - Average 356 36*+ 1+65 312 Median 250 2^6 318 280 - = Destroyed / - Dead / 266 TABLE h Average Coliforms in Millions Days : 6 Age of niss in weeks : 7 • • • 16 • 1 37.5 21.6 13.1 1+ 75, 90.7 ^9.^ 8 12 118 89.5 1^9 2b0 65 1^7.5 53 TABLE 5 Coliform Counts in Millions (Most probable numbers) Serial: Pig no. : no. 1 1+91+ 2 M-95 3 M-96 6 weeks old niss Before : After infection infection,: 1 st dav : M-th dav : 8th dav :12th dav: l6th dav lM-0 2.5 110 k-97 9.5 l*f lM-0 !+5 lifO M-5 25 b5 lM-0 1^5 if5 95 >+5 - / 95 5 513 25 b5 150 160 - 6 51b b5 , ibo 160 117 - 7 515 20 110 / 8 525 11 1^0 250 9 526 1*+ 110 lif - 10 528 \5 2 16 / 11 512 lb- bo 180 - 12 527 11 70 110 - Average 37.5 75 118 89.5 Median 17 57 l*fO 81.0 - s Destroyed / - Dead - P i- table 6 Average Enterococci in Millions Age of Dies in weeks 0 0 Days 1 8 12 : • • 6 • 0 • 7 4 0 0 0 16 68.2 7 8.7 65.6 3^.3 56.2 U-2.9 83.1 59.2 19.6 79 67.5 67.5 55 TABLE 7 Enterococci Counts in Millions (Most probable numbers) • Serial: Pig no. : no. “ '■ 1■ Before 6 weeks old nigs • • , After infection «L •• _ 1st day : Vth dav : 8th dav :12th dav:l6th dav 1 110 15 lifO lifO - >+5 k5 lifO / 2 >+95 3 if96 95 95 ifO if if97 75 if5 if5 if-5 5 513 120 20 110 15 - 6 5lk 25 15 lifO 130 - 7 515 16 2 8 525 25 25 15 - 9 526 lifO 20 i+5 - 10 528 lifO if 11 512 if5- 12 527 2.5 / Iko / 110 8k - 25 15 110 Average 68.2 3^.3 83.1 79 Median 60 22 8if 87 - r Destroyed / = Dead - 6 56 TABLE 8 Average Lactobacilli Counts in Millions Days : 6 Age of oigs in weeks { 7 : • • • 106 13.b 23 ' 69 6.2 3 *+6 3.1 3.2 21 0.2 1 85 b 8 12 16 • 57 TABLE 9 Lactobacilli Counts in Millions — — — 6 weeks old piss After infection _ Serial: Pig no. : no. Before : infection: 1 st dav : *+th dav : 8th dav :12th dav: 16th da.v 1 1+9!+ 55 2 1.5 1.0 - 2 1+95 *+3 6 1.0 12.6 - 3 b 96 56 9.5 8.2 3.5 / if b9 7 1 2.7 0.7 .01 5 513 13 6 3 1.7 - 6 5l*f 105 b.5 3 7 515 80 8 525 31.8 9 526 312 10 528 200 11 512 98 85 12 527 32 127 Average 85 23 Median 68 1.0 / z Dead / .073 .013 - .020 lr\ H O• - - Destroyed 20 .018 - 23. i+ 13 / 5 - .018 - 3 3.2 3 1.3 - 58 TABLE 10 Plate Counts (Aerobic) in Millions Serial; Pig no. : n o . Before : After infection 1st ..day ; >+th da.v : 8th dav :12th davsloth dav 1 610 1380 2060 3850 3900 3950 2 613 350 215 307 312 200 3 6lV 1180 2150 2060 / b 659 267 235 277 155 5 660 238 133 283 / 6 661 265 238 333 - 7 662 313 380 650 - 8 663 211 210 500 317 9 66b- 270 219 18*+ 208 500 10 665 363 315 720 2 57 330 11 666 363 328 311 tf70 306 12 667 236 213 330 295 300 13 668 2^6 305 217 211 630 lb- 501 lH-60 215 82 / 15 503 1110 l1+6 132 - 16 612 168 213 190 - Average 502.6 V73.5 631.6 685.0 802 Median 291 236 216 295 330 - - Destroyed / = Dead - - TA B L E 11 Goliform Counts in Millions (Most probable numbers) Serial: Pig no. : no. 7 weeks old oigs Before : After infection infection: 1st dav : ifth dav : 8th dav :12th dav:l6th dav 1 610 15 35 30 no 250 2 613 20 30 lifO 250 >f5o 3 6n 115 lifO 20 / h 659 if lifO 300 325 5 660 if 200 300 / 6 .661 11 15 112 - 7 662 8 150 160 - 8 663 ^5 110 160 no 9 66if 12 110 lifO 120 200 10 665 if5 30 110 no 150 11 666 *+5 if^ 110 300 if50 12 667 250 if50 if5o 300 13 668 15 20 lifO 300 if50 n 501 25 25 if5 / 15 503 15 11 16 612 30 lifO 7.5 no - - / 3^ / Average 21.6 90.7 lif9 2if0 300 Median 15.0 77.5 no 250 22 7 - = Destroyed / - Dead 60 TABLE 12 Enterococci Counts in Millions (Most probable numbers) 7 weeks old DiKS Serial: Pig Before : After infection n o . : n o . infection: 1st dav : Ifth dav : 8th dav :12th dav:loth dav 1 610 ll+O 2 613 1+5 3 6lb b 659 30 1+0 li+O 5 660 25 16 1+5 / 6 661 l*+0 16 If5 - 7 662 20 11 lb - 8 663 1^0 110 b5 30 9 661+ 30 110 1+5 !+5 10 665 20 25 30 16 11 66 6 110 lifO 150 1^0 250 12 667 110 120 160 110 160 13 668 lifO 15 30 200 150 1*+ 501 l!+0 1+0 110 / 15 503 25 lb 1+5 - 16 612 1+5 20 10 - Average 78.7 56.2 59.2 67.5 Median 77.5 20 1+5 30 no 9.5 - = Destroyed / - Dead 160 l+ *+5 25 30 16 9.5 7.5 ll+ 20 / 11.5 105 1+5 6l TABLE 13 Lactobacilli Count in Millions Serial: Pig no. : n o . 7 weeks old nigs Before s After infection infection: 1st dav : Vth dav : 8th dav :12th dav: l6th dav 1 610 16 .08 .09 .07 .07 2 613 18 .036 .06 .03 .Ob 3 6m 72 .03 ,0b b 659 68 53 38 7 5 660 212 31 93 / 6 661 28 18 7 7 662 68 3 8 663 276 13 8 10 - 9 66!+ 232 2!+0 133 27 7 10 665 66 218 23 25 7 11 666 180 182 193 12 b5 12 667 215 209 206 86 3 13 668 178 55 18 20 0.82 lb 501 6 lb 10 / 15 503 26 6b 8 16 612 !+8 Average Median - - .065 - 106 69 b6 21 10 68 2b 9 10 3 - = Destroyed / - Dead - - .08 0.07 / TABLE lV Total Bacterial Population (in Millions) (8 weeks old pigs before inoculation) • • • Serials Pig :Plate counts Coliforms Enterococci Lactobacilli n o . : no. : (aerobic) 1 V59 165 11.5 2 *+63 163 3 516 V5 6 Vo 1V0 V3 1110 15 V5 122 517 2V0 V5 110 98 5 518 308 IV 160 58 6 6V6 293 15 lVO 50 7 6b7 3lV 20 110 158 8 650 205 25 9 692 158 15 10 693 227 11 695 3 67 Average Median 7.5 17 20 50 15 88 16 1V0 150 322 20 8V 76 227 15 110 58 3.5 63 TABLE 15 Total Bacterial Population (in Millions) (10 weeks old pigs before inoculation) • • • • • • Serials Pig :Plate counts Coliforms Enterococci:Lactobacilli • • no. ; no. : (aerobic) 1 1+61+ 2 980 15 15 3.5 6b-3 if 9 3.0 3 k66 251 20 15 21.6 if b6 7 250 25 Ibo 12.0 Average 521 16 1+5 Median 1+1+7 17.5 15 10 7.5 6*f TABLE 16 Total Bacterial Population (in Millions) (12 weeks old pigs before inoculation) • * • Serial: Pig sPlate counts Coliforms Enterococci Lactobacilli n o . : n o . : (aerobic) 1 ifl7 225 if 2? 16 2 Ifl8 303 5 20 9 3 ^22 b^5 *f ifO 8 b b23 b+ 600 25 l>+0 5 602 *+5 25 20 6 605 20 30 20 7 606 *+5 !+5 15 8 608 30 13 Average 65.6 b2.9 19.6 67.5 Median h-5 27 20 67.5 - - Destroyed / = Dead 110 / - 25 7-5 Constipated No sample r7 TABLE 20 Lactobacilli Counts in Millions Serial: Pig no. : n o . 16 V7eeks old pigs Before : After infection infection: 1 st day : bth dav : 8th dav :12th dav:l6th dav 1 599 8.6 2 603 3 18 / 3 60>+ ^5 5 2 b 600 6 5 - 5 602 , 0.8 0.6 0.3 0.1 6 605 7.8 6. o.b - 7 606 22. • CO - 8 608 1^ 0.8 0.16 Constipated No sample Average 13.b 6.2 3-1 0.2 Median 8 5 1.2 0.2 / = Dead 15 b.5 H - a Destroyed 3.8 0.3 - / / 69 TABLE 21 Total Bacterial Population (in Millions) (19 weeks old pigs before inoculation) • • « • • Serial: Pig sPlate counts Coliforms sEnterococci Lactobacilli • • no. : no. : (aerobic) 1 1&8 1^9 . 7.5 30 5 2 W9 132 25.0 120 2 3 bfO ^fOO 15 16 8 b b$2 200 8 16 10 5 b$b 158 11.5 30 7 Average 208 13 b2 6,b Median 158 11.5 16 7 70 TABLE 22 Mean of the Counts of the Micro-organisms (in Millions) at Different Ages of Pigs on the First Day (Before infection) Total counts : Coliforms : Enterococci : Lactobacilli 6 weeks age 356 37 68 85.6 7 weeks age 502.6 21.6 78.6 106.8 8 weeks age 322 20 8*f 76.1f b-5 10 !+6 13 66 13 .')+ 10 weeks age 521 16 12 weeks age 371 19 16 weeks age 2^+9 13 19 weeks age 208 13 ^2 TABLE 23 Classification Chart Showing Growth of Streptococci in Different Conditions* Division Enterococcus : : Group of species : : Growth : Growth in presence : at i of c s6 .5%i pH :0.1 fo methylene: ;io° c;^^° :I'TaCl: 9.6: blue : • Survival at 60° C, 30 min. S. fecalis / / / / / / S. liauefaciens / / / / / / S. zvinogenes / / / / / r/ / / / / / S . durans *Bact. Rev., 1:16. 1937* 72 TABLE 2h Percentage Distribution of Enterococci Subgroups Name of organism Str. fecalis : Number of cultures: 52 Percent 81.25 Str. durans 8 Str. liauefaciens 3 1+.68 Str. zvmoeenes 1 1.2 12.5 SUMMARY AND CONCLUSIONS In the study of the intestinal flora of swine during health at the ages of six to seven weeks, the highest count was found to be that of lactobacilli, followed by enterococci, with the coliforms being the least numerous. The total counts always exceeded the sum of the group counts. In the pigs ten to nineteen weeks of age, the lacto­ bacilli counts decreased dramatically even during health. Although enterococci counts were quite low after eight weeks of age, they did not become insignificant, and were higher than those of the coliforms and lactobacilli. 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